title: A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity.
abstract: Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA. disom@med.unc.edu
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Isom
authors.forename: Philippe R
authors.initials: PR
authors.lastname: Marguet
authors.forename: Terrence G
authors.initials: TG
authors.lastname: Oas
authors.forename: Homme W
authors.initials: HW
authors.lastname: Hellinga
mesh_terms: D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D011506:Proteins; C028003:RbsB protein, E coli; D008836:Micrococcal Nuclease; D003545:Cysteine